ObjectiveTo systematically investigate the functional roles of the nucleotide metabolism-related gene ecto-5'-nucleotidase (NT5E) in lung adenocarcinoma (LUAD) and to provide potential molecular targets for precision diagnosis and targeted therapy. MethodsMulti-omics analyses were performed using transcriptomic data from The Cancer Genome Atlas (TCGA) and gene expression omnibus (GEO) to identify key prognostic genes and to construct a prognostic model. Tumor tissue specimens from patients with LUAD who underwent surgical resection at the Qingdao Central Hospital of University of Health and Rehabilitation Sciences between May and June 2023 were collected for prognostic gene validation, and tissue microarrays were constructed to evaluate protein expression. In vitro, the biological effects of NT5E on LUAD cells were validated by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, wound-healing, Transwell, EdU, cell-cycle, and apoptosis assays. ResultsA total of 10 paired tumor and adjacent non-tumor tissue samples were used for validation of key prognostic genes, including 4 male and 6 female patients, with a mean age of (58.00±13.06) years. In addition, tissue microarrays comprising 90 patients were analyzed, including 28 male and 62 female patients, with a mean age of (60.07±7.98) years. Bioinformatic screening identified NT5E as a representative prognostic gene that was significantly upregulated in LUAD tissues. Survival analyses demonstrated that high NT5E expression was associated with markedly poorer overall survival and served as an independent risk factor. qRT-PCR and tissue microarray-based immunohistochemistry further confirmed its elevated expression in tumor tissues. Functional experiments showed that NT5E knockdown significantly inhibited cell migration, invasion, and proliferation while promoting apoptosis, whereas NT5E overexpression exerted the opposite effects. ConclusionNT5E markedly promotes LUAD cell proliferation, migration, and invasion and suppresses apoptosis, indicating that NT5E acts as an oncogenic driver in LUAD and may serve as a promising prognostic biomarker and therapeutic target.